G-protein coupled receptors (GPCRs) are expressed throughout the lung, mediating homeostatic, adaptive, and pathogenic events, and are targets for many therapeutic agents. Human airway smooth muscle (HASM) express hundreds of GPCRs regulating contraction, relaxation, immune response, and growth, with direct relevance to asthma and COPD. However, substantial inter-individual variability in function, heterogeneity of signaling, and paradoxical responses of GPCRs are frequently found in clinical, ex vivo, and in vitro studies. Defining the molecular basis of this variability has been the broad long-term goal of this grant, and is critical for understanding disease pathobiology and heterogeneity, development of new drugs, and pharmacogenomics. During the past 5 years, polymorphisms of individual HASM GPCR genes were identified in a reference population and functionally characterized. These studies identified one mechanism of signaling variability in HASM, but also showed that the variability cannot be entirely explained by receptor polymorphisms. Three novel mechanisms of lung signaling variability were uncovered: alternative splicing, heterodimer formation, and genetic variation of G-protein coupled receptor kinases (GRKs). Thus the HASM receptorome is much more complex than previously recognized. In Aim 1, the structural and signaling effects of alternatively spliced GPCRs expressed in HASM will be determined. We have recently found that ~50% of GPCRs express multiple receptor isoforms in HASM due to alternative pre-mRNA splicing, which is subject to inter-individual variation. These isoforms will be cloned, expressed, and characterized in model-cell systems, and the signaling phenotypes confirmed in HASM. In Aim 2, heterodimer formation from selected GPCR pairs relevant to obstructive airway disease will be ascertained and their function determined. We find that HASM GPCRs can form heterodimers, often between very different receptors, that act as distinct signal-transduction units and alter airway responsiveness. Resonance energy transfer techniques will be utilized for detection and characterization of heterodimers, as well as studies of intracellular signaling events related to Gs, Gi, and Gq-coupled receptors in transfected cells and HASM. In Aim 3, the signaling impact of a GRK5 variant will be determined for selected GPCRs relevant to obstructive airway disease. GRKs regulate agonist-promoted function for most GPCRs. We have found a polymorphism of GRK5 that substantially alters 2AR function compared to WT GRK5, thus indicating a mechanism of variability at a hierarchic point above an individual receptor. Selected GPCRs will be studied in recombinant systems and HASM to ascertain the GRK5 variant phenotype in regards to receptor phosphorylation, internalization, desensitization, and GRK/-arrestin signaling. Collectively, results from these studies will define mechanisms of inter-individual variability and heterogeneity of HASM GPCRs, providing a basis for the diversity of phenotypes and drug-responses in obstructive lung disease.